Aqueous lysine-containing animal feed supplements and process for the production thereof

ABSTRACT

The invention relates to aqueous L-lysine-containing animal feed supplements, which optionally contain inactivated microorganisms from fermentation and have a pH value greater than 4 and less than 5 and to a process for producing this preparation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.09/599,644, now U.S. Pat. No. 6,340,486 filed on Jun. 23, 2000. It alsoclaims priority to U.S. provisional application No. 60/140,449, filed onJun. 23, 1999, which is hereby incorporated by reference.

FIELD OF THE INVENTION

The invention relates to aqueous L-lysine-containing animal feedsupplements, which optionally inactivated microorganisms from thefermentation process, and a process for the production of suchsupplements.

BACKGROUND OF THE INVENTION

The essential amino acid L-lysine is widely used as an animal feedsupplement. It is known that L-lysine is produced by fermentation ofstrains of coryneform bacteria, in particular Corynebacteriumglutamicum. Due to its great significance, efforts are constantly beingmade to improve the production process. Improvements to the process mayrelate to the intrinsic performance characteristics of themicroorganisms (e.g., by mutagenesis and selection), to measuresconcerning fermentation technology (e.g., stirring and oxygen supply),to the composition of the nutrient media (e.g., sugar concentrationduring fermentation), or to ways in which product is purified orprepared (e.g., ion exchange chromatography or crystallisation.

According to the prior art, there are three different groups ofL-lysine-containing product presentations which are produced fromfermentation broths. The best known group comprises pulverulent orcrystalline product presentations obtained from cleaned-up or pureL-lysine, which is typically present in the form of a salt such as, forexample, L-lysine monohydrochloride. Another group of solid productpresentations, (see e.g., EP-B-0533039), contain, in addition toL-lysine, the feedstock used during fermentative production and,optionally, the inactivated biomass of the microorganism used. Thesetypes of solid products often exhibit clear disadvantages with regard tohandling properties and application. For example, the quality of theproduct may be influenced to a considerable extent by the weather (highair humidity levels may cause caking which impairs the shelf life anddispensing properties of the product). In addition, the processing ofdry, pulverulent L-lysine-containing products may lead to undesirabledust development.

The third group of L-lysine-containing products comprises concentrated,aqueous, alkaline solutions. These avoid the above-mentioned problems(EP-B-0534865).

Many different processes are known for producing L-lysine-containingproducts from L-lysine-containing fermentation broths. There areessentially two different processes currently known for producing solid,pure L-lysine. Lysine may be obtained as a monohydrochloride (Lys-HCl)by crystallisation from the corresponding fermentation broths, after theinactivated biomass has been separated off by suitable methods.Cleaning-up of the filtrate prior to further concentration is performed,in general, by ion exchange chromatography over several stages.Typically, fermentation broth separated from the biomass is acidified,preferably by the addition of hydrochloric acid (HCl) or sulfuric acid(H₂SO₄), to ease adsorption of the lysine on to ion-exchange resins. Oneproblem with this is that, in addition to L-lysine, various othercations are present in the fermentation broth which are also bound. Theadsorbed lysine is then preferably eluted by an ammoniacal solution andthe ion-exchange column is regenerated. The lysine solution obtained inthis way is then concentrated and lysine-HCl is obtained in crystallineform after neutralisation with hydrochloric acid. In general, a varietyof ion-exchange columns connected in sequence are necessary forobtaining a pure product.

Another method enables lysine to be obtained in the form of acrystalline salt after purifying with activated carbon (SU-183581). Thelysine-containing fermentation broth is inactivated by standardprocesses using moist heat and separated from the biomass by filtration.After acidification of the filtrate to pH 5, 4-5% activated carbon isadded with constant stirring at 50-55° C., in order to separate offundesirable impurities from the filtrate and to prevent discoloration ofthe crystallizate. In a further filtration step, the activated carbon isseparated off and the dissolved sulfate is then precipitated as calciumsulfate by the addition of calcium hydroxide. This is filtered off, theammonia is removed in a rotary evaporator under vacuum and the solutionis concentrated until crystallisation occurs on cooling.

The disadvantage of these two preparation methods lies in the numerousindividual stages and the complex cleaning processes using ion-exchangechromatography. The elimination of troublesome salts or the use ofdifferent elution media creates additional waste streams, which musteither be cleaned up by complex methods or disposed of.

EP-B-0533039 avoids these disadvantages in that all the fermentationfeedstock, optionally including the biomass, is concentrated andspray-dried at elevated temperature.

U.S. Pat. No. 5,990,350 describes a process for producing biomass-freeL-lysine-containing granules. In this process, the biomass is firstisolated from the fermentation broth by ultrafiltration and discarded.The filtrate recovered in this way is then concentrated by extensiveevaporation of the water content. The concentrate obtained in this wayis finally dried and worked up in a complex manner by fluidised bedspray granulation to produce granules.

A process is also known from EP-B-0534865 for producing aqueous, basic,L-lysine-containing solutions from fermentation broths in which biomassis separated from the fermentation broth and discarded. A pH value ofbetween 9 and 11 is established using a base such as sodium, potassiumor ammonium hydroxide. The mineral constituents (inorganic salts) areseparated from the broth by crystallisation after concentration andcooling and either used as fertiliser or discarded (land-filled).

OBJECT OF THE INVENTION

The object of the invention is to provide novel, aqueous preparations ofL-lysine and its salts suitable as a feed supplement. In addition, it isan object of the invention to provide a production process that is moreeconomical and efficient than processes currently known.

DESCRIPTION OF THE INVENTION

The essential amino acid lysine is widely used as an animal feedsupplement. Microorganisms of the genus Corynebacterium aredistinguished by their capacity to secrete high concentrations ofL-lysine into the corresponding fermentation medium over a relativelyshort period. The production processes are generally performed as fedbatch processes. L-lysine produced by fermentation is currentlypredominantly worked up to produce a crystalline product, powder orgranules.

The present invention provides an animal feed supplement derived fromthe fermentation broth of Coryneform bacteria, characterised in that itcontains

a) L-lysine and/or a salt thereof, preferably at a concentration of18-35 weight percent (calculated on the basis of lysine base),

b) the biomass formed during fermentation in an amount of from 0 to100%, preferably from 50 to 100% and more preferably from 90 to 100%,and

c) preferably, greater than 50% of the other dissolved and suspendedconstituents of said fermentation broth.

The animal feed supplement is in the form of an aqueous composition at apH of greater than 4 and less than 5.

The animal feed supplement has a lysine content (as lysine base) of 10wt. % to 35 wt. %, preferably 15 wt. % to 35 wt. %, particularlypreferably 18 wt. % to 35 wt. % and very particularly preferably 21 wt.% to 34 wt. %, relative to the total amount of supplement. The pH isless than 7, preferably 2 to 6.5, particularly preferably 2.5 to 6 andvery particularly greater than 4 and less than 5.

The total solids content of the supplement amounts to from 10 wt. % to55 wt. %, preferably 20 wt. to 55 wt., particularly preferably 35 wt. %to 55 wt. % and very particularly preferably 45 wt. % to 54 wt. %. Thesolids content contains, in addition to lysine preferably the biomass ofthe producing microorganism, the inorganic and other organicconstituents of the fermentation broth and the by-products producedduring fermentation, insofar as they have not been separated off bysuitable processes such as, for example, filtration.

If the biomass is left completely or partially in the supplement, thelatter contains the microorganism protein in a concentration of up to amaximum of 4 wt. %. The supplement contains as inorganic constituents,inter alia, calcium, magnesium, phosphorus in the form of phosphate, andsulfur in the form of sulfate. As organic constituents I contains, interalia, vitamins (e.g., biotin and thiamine) and sugars (e.g.,isomaltose).

The organic by-products formed in small amounts during fermentationinclude L-amino acids selected from the group comprising L-alanine,L-asparagine, L-glutamine, L-methionine, L-threonine and L-valine. Theyadditionally include organic acids which carry one to three carboxylgroups, such as lactic acid, acetic acid and malic acid. Finally, theyalso include sugars, such as trehalose. These compounds may be desirableif they improve the valency of the supplement.

The invention further provides a process for producing an aqueous animalfeed additive (in the form of a solution or suspension) containinglysine or salts of lysine from a fermentation broth comprising:

(a) producing an L-lysine-containing broth by the fermentation ofCoryneform bacteria;

(b) adjusting the pH of the broth to a value of greater than 4 and lessthan 5.; and

(c) concentrating the broth obtained under vacuum;

wherein the order of steps (b) and (c) is optional.

Mutant coryneform bacteria producing L-lysine are described in detail inthe prior art, such as for example in U.S. Pat. No. 4,657,860. For thepurpose of L-lysine production, these strains may be culturedcontinuously or discontinuously using the batch process or the fed batchprocess or the repeated fed batch process. A summary of known culturemethods is given in the textbook by Chmiel (Bioprozesstechnik 1.Einführung in die Bioverfahrenstechnik (Gustav Fischer Verlag,Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren undperiphere Einrichtungen (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).

Examples of suitable fermentation media may be found in patentspecifications EP-B-0 532 867, U.S. Pat. No. 5,840,551 and 5,990,350.The culture medium to be used must satisfy the requirements of theparticular strains. Carbon sources which may be used include sugars andcarbohydrates (e.g., glucose, sucrose, lactose, fructose, maltose,molasses, starch and cellulose) oils and fats (e.g., soya oil, sunfloweroil, peanut oil and coconut oil) fatty acids (e.g., palmitic acid,stearic acid and linoleic acid) alcohols (e.g., glycerol and ethanol)and organic acids (e.g., acetic acid). These substances may be usedindividually or as a mixture. Nitrogen sources which may be used includeorganic, nitrogen-containing compounds such as peptones, yeast extract,meat extract, malt extract, corn steep liquor, soya flour and urea orinorganic compounds such as ammonium sulfate, ammonium chloride,ammonium phosphate, ammonium carbonate and ammonium nitrate. Thenitrogen sources may be used individually or as a mixture. Phosphorussources which may be used include potassium dihydrogen phosphate anddipotassium hydrogen phosphate or the corresponding sodium-containingsalts. The culture medium should additionally contain salts of metals,such as magnesium sulfate or iron sulfate, which are necessary forgrowth. Finally, essential growth-promoting substances, such as aminoacids and vitamins, may be used in addition to the above-mentionedsubstances. Suitable precursors may furthermore be added to the culturemedium. The stated feedstock substances may be added to the culture as asingle batch or be fed appropriately during cultivation.

Basic compounds, such as sodium hydroxide, potassium hydroxide, ammonia,or acidic compounds, such as phosphoric acid or sulfuric acid, are usedto control the pH of the culture. Foaming may be controlled usingantifoaming agents such as fatty acid polyglycol esters. Plasmidstability may be maintained by the addition to the medium of suitableselectively acting substances, for example, antibiotics. Theintroduction of oxygen or oxygen-containing gas mixtures such as airinto the culture and thorough mixing using suitable stirring systems orthe gas stream may be used to maintain aerobic conditions. Thetemperature of the culture is typically 25° C. to 37° C. The culture iscontinued until the maximum quantity of L-lysine has formed. This aim isnormally achieved within 10 to 160 hours.

Analysis of L-lysine may be performed by anion exchange chromatographywith subsequent ninhydrin derivatisation, as described in Spackman etal. (Analytical Chemistry, 30:1190 (1958)) or it may be performed byreversed phase HPLC, as described in Lindroth et al. (AnalyticalChemistry 51:1167-1174 (1979)). The fermentation broths used for theprocess according to the invention preferably have an L-lysine contentgreater than 60 g/L (as lysine base) for a content of non-metabolisedsugar of less than 5.0 g/L. Out of a total solids content of >10 wt. %,the biomass preferably accounts for 1 to 4 wt. %. The content ofby-products and vitamins from fermentation (amino acids, organic acids)is preferably less than 2 wt.

In the process according to the invention, the biomass present in thefermentation broth is generally inactivated or destroyed at the start,for example, by heat treatment. Inactivation may optionally be dispensedwith, however. A pH value of less than 7, preferably 2 to 6.5,particularly preferably 2.5 to 6 and very particularly preferably 2.5 to5 is then established in the fermentation broth by means of an inorganicacid, such as sulfuric acid, hydrochloric acid or phosphoric acid, or anorganic acid, such as citric acid, acetic acid or formic acid, or amixture of different acids. The biomass is optionally separated offcompletely or partially by generally known separation or filtrationmethods before or after acidification. It is not generally necessary toseparate off the mineral constituents. The acidic L-lysine-containingsolution or suspension is then concentrated using known methods (such asfor example a rotary evaporation, film evaporation or falling-filmevaporation), preferably under vacuum, until a liquid product isobtained with a lysine content (as lysine base) of 10 wt. % to 35 wt. %,preferably 15 wt. % to 35 wt. %, particularly preferably 20 wt. % to 35wt. % and very particularly preferably 21 wt. % to 34 wt. %, for a totalsolids content of 10 wt. % to 55 wt. %, preferably 20 wt. % to 55 wt. %,particularly preferably 35 wt. % to 55 wt. % and very particularlypreferably 45 wt. % to 54 wt. %. It is generally the case that the brothis so concentrated that preferably no mineral constituents (inorganicsalts) precipitate out of the fermentation broth and the lysine ispresent in solution. A desired concentration of L-lysine may optionallybe established in the product by the addition of an L-lysine-containingsubstance at any process stage.

The solution/suspension obtained in this way has an acid pH value, iseasy to transport, may be readily dispensed, is microbially stable andhas a longer shelf life than an alkaline solution. The term “suspension”is used to indicate that the preferably inactivated microorganisms arepresent in the product in undissolved form.

Acidification may also be performed after or during concentration. Theanions acting as sulfate, chloride, phosphate, citrate etc. may be addedto the medium in the form of commercially available salts even prior tofermentation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is explained in more detail below with the aid ofpractical examples.

EXAMPLE 1 Production of an L-lysine-Containing Fermentation Broth

Fermentation processes for producing lysine-containing broths, which areworked up according to the invention, have been described in detail invarious prior patents (EP-B 0 532 867 and U.S. Pat. No. 5,840,551).

EXAMPLE 2 Production of a Biomass-Free Product

20 kg of a fermentation broth with a lysine content of approximately 9.5wt. % (as lysine base) were produced in accordance with Example 1. Thebiomass present in the fermentation broth was first inactivated by heattreatment for 30 minutes at 80° C. The inactivated biomass was thenseparated off by centrifuging for 20 minutes at 4000 rpm(Biofuge-Stratos laboratory centrifuge, Heraeus, Düsseldorf, Germany).1.0 L of the supernatant purified in this way was adjusted to a pH valueof approximately 4 by the stepwise addition of concentrated sulfuricacid. The liquid content of the centrifugate was then reduced undervacuum using a rotary evaporator (Büchi Rotavapor RE-120 laboratoryrotary evaporator, Büchi-Labortechnik GmbH, Konstanz, Germany) toapproximately 50% dry matter content.

The liquid product obtained in this way had an L-lysine content of 31.5wt. % (as lysine base) and a pH value of 4. The total solids amounted to49.3 wt. %.

EXAMPLE 3 Production of a Biomass-Containing Product

20 kg of a fermentation broth with a lysine content of approximately 9.5wt. % (as lysine base) were produced in accordance with Example 1. Thebiomass present in the fermentation broth was inactivated directly in abioreactor by heat treatment for 30 minutes at 80° C. 1.0 L of thisbiomass-containing fermentation broth then was adjusted to a pH value ofapproximately 4 by the stepwise addition of concentrated sulfuric acid.The liquid content of this acidic lysine-containing fermentation brothwas then reduced under vacuum using a rotary evaporator (Büchi RotavaporRE-120 laboratory rotary Evaporator, Büchi-Labortechnik GmbH, Konstanz,Germany) to approximately 50% dry matter content.

The liquid product obtained in this way had an L-lysine content of 21.8wt. % (as lysine base) and a pH value of 4. The total solids amounted to50.7 wt. %. No significant change in content was noted after 16 monthsstorage at 20° C.

EXAMPLE 4 Production of a Biomass-Free Product with a pH of 4.5

20 kg of a fermentation broth with a lysine content of approximately 9.5% by weight (as lysine base) was produced in accordance with Example 1.The biomass present in the fermentation broth was first inactivated bythermal treatment for 30 minutes at 80° C. The inactivated biomass wassubsequently separated by centrifugation for 20 minutes at 4000 rpm(laboratory centrifuge Biofuge-Stratos, Heraus, Dusseldorf, Germany).1.0 L of the supernatant clarified in this manner was adjusted by thegradual addition of sulfuric acid to a pH of approximately 4.5. Theliquid component was subsequently reduced to approximately 50% drycontent in the centrifuge on a rotary evaporator (laboratory rotaryevaporator, Büchi Rotavapor, RE-120, Büchi-Labortechnik GmbH, Constance,Germany) under a vacuum.

The liquid product obtained in this manner had an L-lysine content of31.6% by weight (as lysine base) and a pH of 4.5. The total dry mass was49.4% by weight.

EXAMPLE 5 Production of a Product with pH 4.5 Containing Biomass

29 kg of a fermentation broth with a lysine content of approximately9.5% by weight (as lysine base) was produced in accordance withExample 1. The biomass present in the fermentation broth was inactivateddirectly in the bioreactor by thermal treatment for 30 minutes at 80° C.1.0 L of this fermentation broth containing biomass was subsequentlyadjusted by the gradual addition of sulfuric acid to a pH ofapproximately 4.5. The liquid component of this acidic,lysine-containing fermentation broth was subsequently reduced toapproximately 50% dry content in a rotary evaporator laboratory rotaryevaporator, Büchi Rotavapor, RE-120, Büchi-Labortechnik GmbH, Constance,Germany) under a vacuum.

The liquid product obtained in this manner had an L-lysine content of21.9% by weight. After a storage of 16 months at 20° C. no significantchange of the content could be determined.

EXAMPLE 6 Production of a Product with a pH 4.8 Containing Biomass

20 kg of a fermentation broth with a lysine content of approximately9.5% by weight (as lysine base) was produced in accordance withExample 1. The biomass present in the fermentation broth was inactivateddirectly in the bioreactor by thermal treatment for 30 minutes at 80° C.1.0 L of this fermentation broth containing biomass was subsequentlyadjusted by the gradual addition of concentrated sulfuric acid to a pHof approximately 4.8. The liquid component of this acidic,lysine-containing fermentation broth was subsequently reduced toapproximately 50% dry content in a rotary evaporator (laboratory rotaryevaporator, Büchi Rotavapor, RE-120, Büchi-Labortechnik GmbH, Constance,Germany) under a vacuum.

The liquid product obtained in this manner had an L-lysine content of21.9% by weight (as lysine base) and a pH of 4.8. The total dry mass was50.6% by weight. After a storage of 16 months at 20° C., no significantchange of the content could be determined.

What is claimed is:
 1. An animal feed supplement derived from thefermentation broth of Coryneform bacteria, comprising: (a) L-lysine or asalt of L-lysine; and (b) from 0-100% of the biomass formed during thefermentation of said Coryneform bacteria; and and wherein said animalfeed supplement is in the form of an aqueous composition at a pH ofgreater than 4 and less than
 5. 2. The animal feed supplement of claim1, wherein said L-lysine or salt of L-lysine is present at aconcentration of 18-35 weight percent, calculated on the basis of lysinebased.
 3. The animal feed supplement of either claim 1 or claim 2,further comprising greater than 50% of the dissolved and suspendedconstituents of said fermentation broth.
 4. An animal feed supplementderived from the fermentation broth of Coryneform bacteria, comprising;(a) L-lysine or salts of L-lysine at a concentration of 18-35 weightpercent, as lysine based; (b) from 0-100% of the biomass formed duringthe fermentation of said Coryneform bacteria; and (c) greater than 50%of the dissolved and suspended constituents of said fermentation broth;and wherein said animal feed supplement is in the form of an aqueouscomposition at a pH of greater than 4 and less than
 5. 5. The animalfeed supplement of claim 4, wherein said animal feed supplement consistsof: (a) L-lysine or salts of L-lysine at a concentration of 18-35 weightpercent, as lysine base; (b) from 0-100% of the biomass formed duringthe fermentation of said Coryneform bacteria; and (c) greater than 50%of the dissolved and suspended constituents of said fermentation broth;and wherein said animal feed supplement is in the form of an aqueouscomposition at a pH of greater than 4 and less than
 5. 6. The animalfeed supplement of any one of claim 1, 4 or 5, wherein said animal feedsupplement comprises 50-100% of the biomass formed during thefermentation of said Coryneform bacteria.
 7. The animal feed supplementof any one of claim 1, 4 or 5, wherein said animal feed supplementcomprises 90-100% of the biomass formed during the fermentation of saidCoryneform bacteria.
 8. A process of producing an aqueous animal feedadditive containing lysine or salts of lysine from a fermentation brothcomprising the steps of: (a) producing an L-lysine-containing broth bythe fermentation of Coryneform bacteria; (b) adjusting the pH of thebroth to a value of greater than 4 and less than 5; and (c)concentrating the broth obtained under vacuum; wherein the order ofsteps (b) and (c) is optional.
 9. The process of claim 8, furthercomprising separating the biomass from the fermentation broth of step(a).
 10. The process of either claim 8 or 9, further comprisingachieving a desired concentration in said feed additive by the additionof L-lysine.
 11. A method of supplementing animal feed, comprisingadding the animal feed supplement of any one of claim 1, 4 or 5.